The long term aim of the research described in this proposal is to understand the structural basis for the unique substrate specificities of the human cytochrome P450 family 4 enzymes. CYP4A and 4F enzymes play important roles in the thermodynamically disfavored omega-oxidation of endogenous fatty acids such as arachidonic acid and the leukotrienes. CYP4B enzymes, which are the focus of this proposal, are mainly associated with the bioactivation of a diverse array of pro-toxins including carcinogenic aromatic amines and the pneumotoxins, 4-ipomeanol and 3-methylindole, while also maintaining omega-hydroxylase selectivity for fatty acids and alkyl hydrocarbons. A major new finding from the previous granting period is that the mammalian CYP4 families possess a unique active-site structural feature relative to all other P450 isoforms. Specifically, a covalently attached prosthetic heme group that is linked to the I-helix backbone through a ester link with an acidic residue - E31 0 in rabbit CYP4B1. This residue appears within a highly conserved I-helix motif -FEGHDT in the CYP4 family of enzymes, although interestingly, several CYP4F enzymes lack this critical acid and appear to exhibit extended substrate specificities. Consequently, a central working hypothesis is that covalent heme binding among CYP4 enzymes restricts conformational flexibility and is a major contributor to their omega-specificity. Therefore, the current competing renewal seeks to fully characterize this novel structural element and ascertain among CYP4 proteins; I) the precise nature of the covalent link to the heme prosthetic group, II) the functional consequences of covalent heme binding, and Ill) the mechanism of formation of the covalent link. I) will be accomplished through 1-D and 2-D 1H-NMR studies on the polar heme species released from covalently bound CYP4 isoforms in order to identify the site of oxygen incorporation into the prosthetic heme group. II) will utilize site-directed mutagenesis to engineer removal of the heme covalent link from CYP4B and insertion of a covalent link into a CYP4F enzyme that lacks the critical I-helix acid in order to evaluate changes in covalent heme binding, substrate specificity and bioactivation. Finally, Ill) will use mass spectrometry and stable-isotope methodology to determine if the mechanism for covalent heme binding to CYP4 involves a carbocation intermediate and the P450-peroxy species.